ES2688284T3 - Dispersion devices for aggregates - Google Patents

Abstract

A continuous flow dispersion device for dispersion of aggregates, the device comprising an upstream entrance at one end of a conduit, at least two obstacles having opposite surfaces, said obstacles being a first or upstream entry obstacle disposed within the conduit, said upstream entry obstacle being perforated by at least one transverse hole and obstructing from about 50% to about 99.9% of the internal cross-sectional area of the conduit, a second or downstream exit obstacle disposed within the conduit and being a downstream exit obstacle drilled by at least one transverse hole and obstructing from approximately 50% to approximately 99.9% of the internal cross-sectional area of the conduit, where the longitudinal axis of any transverse orifice of any of obstacles does not align with the longitudinal axis of any other hole After an anterior or posterior obstacle, a turbulence chamber defined by the opposite surfaces of the first and second obstacles and the internal wall of the conduit between said obstacles, and where the distance between two successive obstacles is approximately 0.1 to approximately 10 times the diameter of the smallest hole of any of two successive obstacles, and an outlet downstream at the opposite end of the duct; and wherein the entrance to the device conduit and the upstream entry obstacle form a first portion of the device, and the exit of the device conduit and the downstream exit obstacle form a second portion of the device and wherein the first and first portions second, they are separable, wherein the device further comprises a spacer seal; wherein, in addition, the conduit in each of the first and second portions comprises a longitudinal light, in which the first and second portions can be juxtaposed and secured together so that a turbulence chamber is defined by the at least two obstacles and at least one inner wall of the spacer seal.

Description

DESCRIPTION

Dispersion devices for aggregates 5 FIELD OF THE INVENTION

[0001] The present invention relates to continuous flow dispersion devices for the dispersion of

aggregates, especially from culture suspensions containing cell aggregates. The present invention provides continuous flow methods for dispersing aggregates to release individual cells. The present invention further relates to continuous flow processes for the homogenization of animal cell suspensions containing cell aggregates.

BACKGROUND OF THE INVENTION

15 [0002] Over the past 100 years, cell cultures have resulted in many applications in the

biotechnology field. The progress in cell culture, especially to obtain a higher cellular productivity, has allowed the development of new processes for the production of recombinant proteins and vaccines, biomass and new uses of cells as cell therapies.

[0003] Cells are grown primarily for two applications. First, the amplification of

Cells through subculture will lead to increases in viable cell biomass production. These viable cells can be used for for example cell therapies, for viral infections and to obtain infected cells and the like. The second application is to produce and isolate biological compounds of interest usually, but not always, present inside the cells, such as polynucleotides, proteins, animal pathogens, or fragments thereof. These bioproducts can also be incorporated into other products such as DNA vaccines, subunit vaccines, viral vaccines, gene therapy compositions, drugs and the like.

[0004] A major problem of cell culture is the formation of aggregates of cells formed during

30 the crop. Cell aggregates, by limiting cell access to nutrients and by inhibiting

contact growth, reduce crop yield in terms of biomass production and

compounds of interest In addition, cell aggregation increases cell death, mainly due to apoptosis. For the production of biomass the collected cells must not be dead or dying to make possible an optimal subculture.

35

[0005] Cells grow attached to a surface (ie anchor dependent) or in

suspension (ie, anchor independent). Most animal cell cultures are anchorage dependent and grow in single-celled layers (monolayers) or on the surface of microcarriers, in plates or flasks. Rolling bottle technology was developed to grow a greater number of animal cells dependent on

40 anchor (Gey G. O., AM.J. Cancer, 17: 752-756 (1933)) although a further improvement arose from the use of

microcarriers in bioreactors, which allows an increase in the area of growth available for cells per unit volume (van Wezel A. L., Nature, 216: 64-65 (1967)).

[0006] These technologies have been used for more than 20 years in the pharmaceutical and medical fields for processes such as cell growth and infection, vaccine preparation, expression of

recombinant proteins, and plant cell culture. Many of these techniques have been published and are used routinely (See for example Freshney, R. I. Culture of animal cells: a manual of basic techniques: 3rd edition 1994).

50 [0007] Usually during the culture of anchor-dependent cells, when the culture reaches a

confluence, it is desirable to disaggregate the culture into individual cells that preserve viability. Cultures of independent anchor cells also show cell groups, and that the problem of dispersion of cell groups exists, regardless of what type of anchor the cells have. The resulting disaggregated suspension can then be subcultured or used directly as a source of a pharmaceutically acceptable compound. Cell dispersion may be a solution to the problems inherent in cell aggregation but it is also problematic, however, due to the fragility of the cells that leads to stress and death.

[0008] Cells are often so well attached to the surface of the underlying culture vessel that

proteolytic enzymes (such as trypsin, collagenase, pronase), chelating agents (such as ethylenediaminetetraacetic acid) and mechanical forces (such as scraping) (Lloyd et al., J. Cell Sci. 22: 671-684 ( 1976); Whur et al., J. Cell Sci. 23: 193-209 (1977); Freyer and Sutherland, Cancer Res. 40: 3956-3965 (1980); Lydersen et al., Bio / Technol. 1: 63 -67 (1985)). The dispersion of aggregates was also tested with deoxyribonuclease (Jordan et al.

5 Animal Cell Technology: Developments, Processes and Products, editorial editors: Spier et al., 418-420 (1992), publishing house: Butterworth-Heinemann, Oxford; Renner et al., Biotechnol. Bioeng., 41: 188-193 (1993),) or with hypoosmolar medium (Leibovitz et al., Int. J. Cell Cloning, 1: 478-485 (1983)). All these treatments are usually insufficient separately to obtain a uniform dispersion of viable individual cells. Normally some groups of visible cells remain with the microscope and / or the naked eye. A

The aggregate or group of cells is a mass of variable size, sometimes visible to the naked eye, formed by the union of individual cells with each other or by the union of cells to at least one other material (ie, remains, extracellular matrix) present in the initial cell suspension. By definition, an aggregate of cells has a minimum size of about 800 pm, in particular a minimum size of about 600 pm, particularly about 400 pm, preferably about 200 pm, more preferably of

15 approximately 100 pm.

[0009] Detached and dispersed cell suspensions may also need to undergo various downstream treatments, for example to remove chemical compounds used during cell collection, such as trypsin. These stages require a lot of time and increase the cost of the product and can give

20 place to an unwanted reintegration. For example, a centrifugation step can be carried out to remove unwanted chemical compounds. This process, however, leads to the formation of a supernatant that contains the chemical compounds and will be discarded, and a sediment comprising cells to collect. When compacted in a sediment, the cells are so close and tight together that aggregates of cells are formed.

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[0010] Compared to microorganisms such as viruses and bacteria, eukaryotic cells, and especially animal cells, are very fragile and shear sensitive due to the lack of a durable cell wall. Shear sensitivity is also related to the type of cell (i.e., if they are fibroblasts, lung cells, kidney cells, etc.), the age and history of the crop (the old cultures that

30 have a high number of steps containing more fragile cells) and maintenance conditions (variations in culture conditions, such as temperature, osmotic pressure, etc. generate tensions). Virus infection can also lead to increased shear sensitivity of infected cells.

[0011] In mouse and human cell culture experiments, wall shear stresses

35 of 100 N / m2 for a residence time of 0.5 seconds cause a cell death rate

significant. Studies on embryonic kidney cells showed that shear stresses of less than 0.26 N / m2 caused a slight reduction in viability and no change in cell morphology (Harbor et al., Adv. Biochem. Eng., Vol. 29. Publishing house: Springer-Verlag (New York), (1984)).

40 [0012] As a general consideration, therefore, the shear forces applied on a

Cell suspension could lead to a decrease in cell viability. Shear forces can decrease the performance of viable cells and can also reduce the ability of cells to divide by inhibiting cell mitosis.

[0013] Since a good cell viability is preferred for pharmaceutical use, a procedure is needed

gentle to disperse a cell suspension that contains cell aggregates. The technology used must be efficient to release individual cells at high production yields, but it also has to be smooth enough to avoid a significant reduction in viability.

[0014] Known methods of cell culture manipulation may involve dispersion with

soft procedures, for example with soft pipetting (ECACC Handbook, Fundamental Techniques in Cell Culture. A Laboratory Handbook, "Protocol 5 - Subculture of suspension cell lines", 2005, edited by Sigma-Aldrich). Pipetting is usually carried out manually by repeated aspiration and rejection of the cell suspension until all cell groups have disappeared. This manual operation is not without

55 However, consistent or reproducible. Different results can be observed using the same starting material of the cell culture from one pipette to another, or from one operator to another. In addition, shear damage is a function of both shear time and shear forces. Pipetting too energetic and / or for too long a period can damage the cells and lead to low viability. Alternatively, pipette too softly or inconsistently and the difficulty of determining when the groups have disappeared

cells can lead to poor cell performance since the remaining cell aggregates will be discarded during subsequent filtration stages. In addition to this lack of robustness, the soft pipetting technique is tedious and requires open phases that increase the risks of contamination. Pipetting does not lend itself to high volume processing. Application EP-590504-A describes a disaggregation device with two 5 cutting plate elements that have perforations and a tubular chamber. Therefore, there remains a need for large-scale processes for the dispersion of cell aggregates, and preferably performed in a closed system to avoid contamination risks. The present invention addresses these problems by providing a continuous flow dispersion device for dispersion of shear-sensitive aggregates, especially suspensions of cultures containing cell aggregates, while respecting the integrity of individual cells and continuous flow procedures for Disperse aggregates of shear sensitive cells to release individual cells.

SUMMARY OF THE INVENTION

The present invention encompasses a continuous flow dispersion device for the dispersion of

aggregates of cells, the device comprising an upstream entry at one end of a duct, a first entry obstacle or upstream into the duct, this upstream entry obstacle having at least one transverse hole providing from about 50% to approximately 99.9% obstruction of the internal cross-section of the conduit, a second or downstream exit obstacle within the interior of the conduit, this downstream exit obstacle having at least one transverse orifice providing from approximately 50% up to approximately 99.9% obstruction of the internal cross-section of the conduit, where the longitudinal axis of any hole through any obstacle does not align with the longitudinal axis of any hole of an anterior or posterior obstacle, and where the distance between two successive obstacles is about 0.1 to about 10 times the d hole diameter smaller than either of two successive obstacles, and an outlet downstream at the other end of the duct; and wherein the entrance to the device conduit and the upstream entry obstacle form a first portion of the device, and the exit of the device conduit and the downstream exit obstacle form a second portion of the device and wherein the first and first portions second, they are separable, wherein the device further comprises a spacer seal; wherein, in addition, the conduit in each of the first and second portions may be juxtaposed and secured to each other such that a turbulence chamber is defined by the at least two obstacles and at least one inner wall of the spacer seal. The various embodiments of the device according to the invention encompass, but are not limited to, a first obstacle inside the conduit that can be perpendicular to the direction of the flow flowing through this conduit, this first obstacle having a plurality of holes that they can be of any configuration including, but not limited to, circular, ovoid, concentric, rectilinear and parallel to the longitudinal axis of the duct, and which provide an obstruction of about 50% to about 99.9% of the duct section , and at least a second obstacle, also inside the duct and perpendicular to the direction of the flow flowing through this duct. This second obstacle can also have a plurality of holes that can be of any configuration including, but not limited to, circular, concentric, rectilinear and parallel to the longitudinal axis of the conduit that create an obstruction of approximately 50% to approximately 99, 9% of the duct section. The holes of the second obstacle are not placed in the same longitudinal alignment as any hole of the first obstacle, and the distance between the first and second obstacles is about 0.1 to about 10 times the diameter of a hole.

[0016] In an advantageous embodiment of the present invention, the device may comprise an input

upstream at one end of a cylindrical shaped conduit, a first obstacle and a second obstacle inside the conduit and perpendicular to the direction of the flow flowing through this conduit, each obstacle having 3 transverse holes whose longitudinal axes are parallel to the longitudinal axis of the duct, and which create approximately 98% obstruction of the duct section, the longitudinal axes of the 50 holes of the second obstacle not being in the same longitudinal alignment as the longitudinal axis of any hole of the first obstacle, in where the distance between the two obstacles is approximately twice the diameter of the smallest hole of any of the obstacles, and an outlet downstream at the other end of the duct.

[0017] The present invention further provides a continuous flow process for the dispersion of

Aggregates, comprising the steps of (1) flow of a suspension containing cell aggregates through a dispersion device according to the invention, thereby disintegrating the aggregates, optionally repeating step (1) by re-flowing the suspension through the device or by serially placing more than one of the continuous flow dispersion devices according to the invention, and (4) collecting the suspension containing individual cells.

[0018] A further object of the invention is to provide a cell culture method, which

comprise the steps of (1) introducing cells to be cultured in a culture batch filled with a culture medium and culturing the cells, (2) flowing the suspension containing cell aggregates obtained in step (1) through 5 at least one continuous flow dispersion device according to the invention, thereby disintegrating the aggregates, (3) reintroducing the suspension obtained in step (2) and containing individual cells in a cell culture batch, (4) optionally repeat steps (1) to (3), and (5) collect the suspension containing individual cells.

[0019] These and other embodiments are described or obvious by and are encompassed by, the following

Detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following detailed description, given by way of example, and not intended to limit the invention to

specific embodiments described, can be understood in conjunction with the attached figures, incorporated in this application by way of reference, in which:

Fig. 1A illustrates a longitudinal sectional view of a dispersion device comprising two portions of conduit 20 connected by a seal, and having unique perforated entry and exit obstacles and a single turbulence chamber. The direction of fluid flow through the device is indicated by the thick arrow.

Fig. 1B illustrates a longitudinal sectional view of a dispersion device comprising two conduit portions connected by a seal and a securing means.

Fig. 2 illustrates a longitudinal sectional view of an embodiment of the continuous flow dispersion device 25 comprising two conduit portions connected by a seal, and having unique perforated entry and exit obstacles and a single turbulence chamber, where the obstacles are removable plates. The direction of fluid flow through the device is indicated by the thick arrow.

Fig. 3A illustrates a front view of an entry obstacle.

Fig. 3B illustrates a front view of an exit obstacle, where the transverse holes are not concentric.

Fig. 4A illustrates a longitudinal sectional view of an embodiment of the continuous flow dispersion device comprising two sequential turbulence chambers.

Fig. 4B illustrates a longitudinal sectional view of an embodiment of the continuous flow dispersion device comprising two sequential chambers and a securing means.

Fig. 4c illustrates a longitudinal sectional view of an embodiment of the continuous flow dispersion device comprising a single unit having two integral turbulence chambers.

DETAILED DESCRIPTION OF THE INVENTION

[0021] It is noted that in this description and particularly in the claims, terms such as

"comprises", "understood", "understood", "comprising", "comprising", "comprising" and the like may have the meaning attributed thereto in the United States Patent Law; p. eg, they can mean "includes", "included", "included", "including", "including", "including", and the like; and that terms such as "consisting essentially of", "consisting essentially of", "consisting essentially of", and "consisting essentially of" have the meaning assigned to them in United States Patent Law, p. For example, they allow elements not explicitly listed, but exclude elements that are found in the prior art or that affect a basic or novel feature of the invention.

[0022] The present invention encompasses a continuous flow dispersion device for the dispersion of 50 aggregates, especially suspensions of cultures containing cell aggregates. It is contemplated that the

device, as illustrated for example, in Figs. 1A-2 and 4A-C, be placed in series with an outflow of a medium / cell suspension from a cell culture system, in which the cell suspension can be passed through a conduit 1 of the device and therefore through a turbulence chamber 2. The cell suspension will be introduced into the continuous flow dispersion device through an upstream inlet 3 of the duct 1 and out of the device through a downstream outlet 4 duct 1.

[0023] The inner walls 10, 11 of the inlet 3 and the outlet 4 of the duct 1 may have smaller cross-sectional diameters than, equal to or larger than that of the turbulence chamber 2. A cross section of an inlet 3 or outlet 4 of the duct is by definition a section perpendicular to the direction of a

flow of liquid passing through conduit 1.

[0024] There is no restriction as to a particular shape of the conduit 1, in particular in the shapes of its cross section. For example the cross section of the conduit 1 may be, but is not limited to, a

5 circular, ovoid, square or rectangular cross section. The cross section of the turbulence chamber 2 is advantageously configured, but not necessarily, according to the shape of the conduit 1.

[0025] The turbulence chamber 2 of the continuous flow dispersion device is defined by the opposite internal walls of two obstacles 14, 15 respectively, a nearest upstream entry obstacle 14

10 to the upstream entrance 3 of the conduit 1, and a downstream exit obstacle 15 closest to the downstream exit 4 of the conduit 1, and an internal wall 16 defining the distance that separates the obstacles from each other. Each obstacle 14, 15, therefore, is located within the conduit 1 and is oriented in the most advantageous way perpendicular to the central axis of the continuous flow dispersion device so as to impede the flow of the cell culture fluid passing through of the duct 1. It is also contemplated that there may be more than two obstacles

15 as shown, for example, in Figs. 4A-4C, wherein each adjacent pair of obstacles and the inner wall of the duct each defines a turbulence chamber 2, thereby forming a plurality of chambers 2 arranged in series. It is further contemplated that the exit obstacle 15 of a first turbulence chamber 2 'can also function as the entrance obstacle for a second turbulence chamber 2' 'located in series immediately downstream of the first chamber 2' as shown in Figs. 4A-4C.

twenty

[0026] Each upstream entry obstacle 14 and a downstream exit obstacle 15 is perforated with at least one transverse hole 16 allowing communication of the entrance 3 and exit 4 of the conduit with the turbulence chamber 2. For the device of continuous flow dispersion of the invention, a transverse hole is defined by an equivalent diameter of a cylindrical shaped hole having the same section. This

25 equivalent diameter advantageously is at least 25 times larger, more advantageously 50 times larger, and more advantageously at least one hundred times larger than the diameter of an individual living cell of the culture.

[0027] The cross-sectional area or combined cross-sectional areas of the hole (s)

30 (s) 16 of an obstacle 14, 15 hinders the flow of fluid through conduit 1 between

approximately 50% to approximately 99.9%. The obstruction of the fluid flow, expressed in a percentage, is calculated by the following formula (XY) / X, where X is the cross-sectional area of the conduit and Y is the sum of hole areas or plurality of obstacle holes . Advantageously, the obstruction of the fluid flow created by an obstacle is approximately 50% to approximately

35 99.9%. More advantageously, the obstruction of the fluid flow created by the obstacle can be from about 60% to about 99.9%, more advantageously from about 70% to about 99.9%, even more more advantageous from about 80% to about 99.9%, and most advantageously from about 90% to about 99.9%.

[0028] No transverse hole 16 of an upstream entry obstacle 14 can be concentric with

with respect to any hole 16 of the downstream downstream exit obstacle 15, ie the longitudinal axis of a hole 16 is not coincident with the longitudinal axis of any other hole of the continuous flow dispersion device as shown, for example, in Figs. 3A and 3B.

[0029] The configurations of the upstream entry obstacle 14 and the downstream exit obstacles

15 and the transverse holes 16 therein, are not limited in terms of the thickness of the obstacles that may also be, but not limited to, a circular, ovoid, square or rectangular cross section, nor in the number, sizes and shapes of the transverse holes 16. The transverse holes 16 may also not be uniform and may have a variety of cross sections. The device can, for example, have each

50 one of all your obstacles with a hole. The device can have all its obstacles 14, 15 with several holes 16 having all the same size and shape. Alternatively, the device may have obstacles 14, 15 with several holes 16 having different sizes and shapes, or each obstacle 14, 15 has the same type of holes but the size and shape of the holes differ from one obstacle to the other.

[0030] It is contemplated that the continuous flow dispersion device of the invention may comprise the

minus two separable portions, an inlet portion 3 and an outlet portion 4 with, as shown for example in Fig. 1A, an optional spacer seal 5 to prevent fluid leakage from the turbulence chamber 2 and to contribute to the separation between the two opposite obstacles 14, 15. Alternatively, as shown in Fig. 2, the obstacles 14, 15 can be independent and removable from the conduit 1 after the separation of

the two portions 3, 4. In another embodiment the device is a single integrated unit, as illustrated, for example, in Fig. 4C. It is further contemplated that the continuous flow dispersion device may further comprise a securing means 6, as schematically illustrated in Figs. 1B and 4B for example, to ensure that the two portions 3, 4 of the device and optionally the spacer seal 5 are not operably connected 5 with any leakage of fluids that pass through the device. The means of assurance may be, but not limited to, a clamp, opposing springs, an elastic seal and the like.

[0031] One aspect of the invention, therefore, encompasses a continuous flow dispersion device for dispersion of aggregates, the device comprising an upstream inlet 3 at one end of a

10 conduit 1, a first or upstream entry obstacle 14 within conduit 1, this upstream entry obstacle 14 having at least one transverse hole 16 that provides an obstruction of about 50% to about 99.9% of the internal cross section of the conduit 1, a second or downstream exit obstacle 15 inside the conduit 1, this downstream exit obstacle 15 having at least one transverse hole 16 that provides an obstruction of about 50% to about 99 , 9% of the

15 internal cross section of the conduit 1, where the longitudinal axis of any hole 16 that pierces any obstacle 14, 15 does not align with the longitudinal axis of any hole of an anterior or posterior obstacle, and where the distance between two successive obstacles it is about 0.1 to about 10 times the diameter of the smallest hole of any of two successive obstacles, and an outlet downstream 4 at the opposite end of the conduit 1.

twenty

[0032] In various embodiments of the continuous flow dispersion device of the invention, the first, or upstream, entry obstacle 14 comprises a plurality of transverse holes 16, wherein the cross-sectional configurations of the holes 16 can be select from circular, concentric and rectilinear, and where the longitudinal axis of each hole 16 is parallel to the longitudinal axis of the conduit, being created from

25 thus an obstruction of approximately 50% to approximately 99.9% of the section of the conduit 1, and wherein the holes 16 may be identical or different from each other.

[0033] In embodiments of the continuous flow dispersion device of the invention, the second, or downstream, exit obstacle 15 comprises a plurality of transverse holes 16, wherein the

30 cross-sectional configurations of the holes 16 are selected from circular, concentric and rectilinear, and wherein the longitudinal axis of each hole is parallel to the longitudinal axis of the conduit 1, thereby creating an obstruction of approximately 50% at approximately 99.9% of the section of the conduit 1, and wherein the holes 16 may be identical or different from each other.

[0034] In an advantageous embodiment of the continuous flow dispersion device of the invention, the

First, or upstream, entry obstacle 14, as shown in Fig. 3A, comprises three transverse holes 16, wherein the cross-sectional configurations of the holes 16 can be selected from circular, concentric and rectilinear, and in where the longitudinal axis of each hole is parallel to the longitudinal axis of the conduit, thereby creating an obstruction of approximately 50% to approximately 99.9% of

40 the section of the duct, and wherein the holes 16 may be identical or different from each other, and the second exit obstacle, or downstream 15 as shown in Fig. 3B comprises three transverse holes 16, wherein the cross-sectional configurations of the holes are selected from circular, concentric and rectilinear, and where the longitudinal axis of each hole is parallel to the longitudinal axis of the conduit, thereby creating an obstruction of approximately 50% to approximately 99.9% of the section

45 of the conduit, and wherein the holes may be identical or different from each other and where the longitudinal axis of any hole 16 through the first obstacle 14 does not align with the longitudinal axis of any hole of the second obstacle 15.

[0035] In the various embodiments of the continuous flow dispersion device of the invention, the

50 upstream entry obstacle 14 and downstream exit obstacles 15 are advantageously, but not

necessarily, perpendicular to the direction of the flow passing through conduit 1.

[0036] In various embodiments of the continuous flow dispersion device of the invention, the holes through the entry obstacle 14 and the downstream exit obstacle 15 can be cylindrical in shape,

55 concentric, or rectilinear, where the longitudinal axes of the holes are parallel to the longitudinal axis of the conduit. A device according to the invention, as shown in the longitudinal section in Fig. 1B, comprises a first portion 3 and a second 4, wherein each of said first portions 3 and second 4 comprises a conduit 1 having a longitudinal light 7 and an obstacle 14, 15 located therein and perpendicular to the longitudinal axis of the light 7 of the conduit 1 and a spacer seal 5, in which the first 3 and second portions 4 can

be juxtaposed and secured together so that a turbulence chamber is defined by the two obstacles 14, 15, and at least the inner wall 17 of the spacer seal 5. (See Example 1, below, and Fig. 1B ).

[0037] Another aspect of the present invention encompasses a method of using the dispersion device of

continuous flow according to the present invention. In particular, the present invention encompasses the use of the device according to the present invention to disperse aggregates, especially suspensions of cultures containing cell aggregates. The method for dispersion of aggregates according to the invention comprises the steps of (1) flowing a suspension containing cell aggregates through at least one continuous flow dispersion device 10 according to the invention, (2) disintegrating the aggregates, (3) optionally repeat step (1) by re-passing the cell suspension through the device, and (4) collecting the suspension containing individual cells. A method for dispersion of aggregates comprises the steps of (1) flow of a suspension containing aggregates of cells through at least one dispersion device, said device comprising an upstream inlet at a tip of a cylindrical shaped duct; a first obstacle inside the conduit and perpendicular to the direction of the flow flowing through this conduit, this first obstacle having three transverse holes, parallel to the longitudinal axis of the conduit, and where the combined cross-sectional area of the holes is approximately 2% of the total cross-sectional area of the duct; a second obstacle downstream inside the outlet conduit and perpendicular to the direction of the flow flowing through this conduit, this second obstacle having 3 transverse holes parallel to the longitudinal axis 20 of the conduit, and where the cross-sectional area combined of the holes is approximately 2% of the total cross-sectional area of the conduit, and where the holes of the downstream exit obstacle are positioned relative to the holes of the upstream obstacle so that they do not have the same longitudinal alignment as no hole of the obstacle upstream, and where the distance between these two obstacles is approximately twice the diameter of a hole, and an outlet downstream at the other end of the duct, 25 (2) disjoint the aggregates, (3) optionally repeat step (1) by re-flowing the suspension through the device (s), (4) collect the suspension q It contains individual cells.

[0038] It is also contemplated that in one embodiment of the process of the invention a

plurality of the continuous flow dispersion devices of the invention for dispersion of aggregates, the devices being placed in series and the suspension containing cell aggregates flowing through all the devices placed in series. For example, two to seven devices can be placed in series or a single device with multiple successive turbulence chambers can be used. A dispersion device can be used in a recirculation mode. The cell suspension container is connected to the inlet upstream of the duct and to the outlet downstream of the duct. The cell suspension flows successively and continuously through the inlet upstream of the conduit, the turbulence chamber, the outlet downstream of the conduit and is recycled inside the cell suspension container. Knowing the processing flow rate in liters per hour through the dispersion device, the volume of the cell suspension in liters, and the total processing time, it is possible to obtain an average number of steps by multiplying the flow rate by Total processing time and then divide the resulting value by the volume of the cell suspension. Three to five dispersion devices can be placed in series, and in a more advantageous embodiment, five dispersion devices are placed in series. Another aspect of the invention is a cell culture method, comprising the steps of (1) introducing cells to be cultured in a culture medium and culturing the cells, (2) displacing or suspending the cultured cells in a medium, wherein the suspension comprises cell aggregates, (3) passing the cell suspension containing cell aggregates through a continuous flow dispersion device 45 according to the invention or through a serial arrangement of such devices, thereby disintegrating the aggregates of cells and releasing individual cells thereof, (4) optionally repeating steps (1) to (3) are repeated as many times as necessary, and (5) reintroducing at least a portion of the disjointed cell suspension of step (3) to the culture batch and reculture the cells.

[0039] In an embodiment of this aspect of the invention, the culture medium may comprise

microcarriers

[0040] In the embodiments of this aspect of the invention, it is contemplated that the continuous flow dispersion device of the invention can be used in a continuous loop, in which the culture medium is circulated to

Through the device to dissociate the groups of cells as they form.

[0041] The devices and methods of the present invention allow, in a continuous flow mode, to subject a cell suspension containing cell aggregates to fluid turbulence to disperse and disintegrate the aggregates thereby freeing individual living cells. The device is a passive device

and does not contain any moving parts, such as a rotor or piston, thereby allowing easy cleaning and reducing the likelihood of damage to the cells.

[0042] Turbulence forces are applied to aggregates during the acceleration of the fluid through the obstacle hole (s) that cross the obstacles and during the redirection of the flow between two obstacles

successive The distance between two successive obstacles and the fact that the hole (s) of the first obstacle is not placed in the same longitudinal alignment as no hole of the next obstacle creates turbulence. In addition, aggregates, and more specifically macroscopic aggregates, are likely to collide with obstacles, which could also favor the dispersion of aggregates.

10

[0043] The continuous flow mode of the present invention compared to a batch mode (ie the pipetting method) has the advantage of avoiding dead spaces inside the turbulence system. All cells pass through the turbulence chamber for efficient dispersion of the aggregates, and as a consequence the continuous flow mode of the present invention results in an improved dispersion of the

15 aggregates. The device of the present invention also allows reducing the open phases by operating in continuous flow mode, which results in a decrease in the risk of contamination.

[0044] Compared to a rotor / stator agitator and the high shear condition, the mode of

Continuous flow of the present invention limits the aggregates to a single and rapid passage inside the device.

20 Due to the flow, there is no possibility for the aggregate to pass twice through the holes of the obstacles, and as a consequence the cells are less subject to tension and the cell viability is better. The processing flow rate will be adjusted to the geometry of the device and the cell type since the shear sensitivity of the cells is related to the cell type, age and culture history and maintenance conditions. Virus infection usually leads to increased shear sensitivity.

25

[0045] Using a hemocytometer and a microscope, the density of a cell suspension can be

establish easily and quickly as well as determine the presence or not of cell aggregates. He

Hemocytometer can also be used to distinguish living cells from dead cells in order to determine the percentage of viable cells. For this purpose it is possible to use vital staining indicators, which only

30 stain non-vital tissues and cells. Cells are permeable to such indicators but living cells are able to exclude them. Dead or dying cells cannot exclude the indicators and therefore have staining. The most common vital staining indicator is trypan blue (Hoffmeister ER, Stain Technol., 28 (6): 309-310 (1953); Boedijn KB, Stain Technol., 31 (3): 115-116 (1956); Allison DC et al., J. Histochem. Cytochem., 28 (7): 700-703 (1980)). By combining such coloration and cell counting techniques it is possible to easily evaluate the percentage of 35 viable cells in a cell suspension, calculated as the number of living cells / (number of dead cells + number of living cells) X 100. It is also It is possible to obtain the same information by flow cytometry using agents that intercalate between nucleic acids such as propidium iodide or 7-a-aminoactinomycin D (7-AAD) (Wattre P., Ann. Biol. Clin. (Paris), 51 ( 1): 1-6 (1993); Lecoeur H. et al., J. Immunol. Methods, 265 (1-2): 81-96 (2002)).

40

[0046] Knowing the percentage of viable cells in a cell suspension, it is easy to determine the optimal conditions that allow obtaining an effective cell dispersion while respecting the viability of the individual cells. Someone skilled in the art will be able to determine the optimal flow rate and determine the optimal number of obstacles in the device or the optimal number of devices to be placed in series to obtain

45 the desired result, that is to say an effective dispersion while maintaining the integrity and viability of the individual cells. Ideally, the average speed of the suspension containing cell aggregates through the holes of the device according to the present invention should be between about 0.1 and about 100 m / s, preferably between about 0.1 and about 20 m / s and more preferably between about 0.5 and about 5 m / s. The cell culture procedures may comprise the 50 steps of (1) introducing cells to be cultured in a culture batch filled with a culture medium and culturing the cells, (2) flow of the suspension obtained in step (1) and which it contains aggregates of cells through at least one device according to the invention and disintegrates the aggregates, (3) reintroducing the suspension obtained in step (2) and containing individual cells in the culture batch, (4) optionally repeat steps (1) to (3), and (5) collect the suspension containing individual cells obtained after stage (2) or stage (4).

55

[0047] This can also be done with microcarrier culture, that is to say that the culture suspension containing cell / microcarrier aggregates passes through at least one device according to the invention. During the dispersion of cell / microcarrier aggregates, the use of the dispersion device according to the invention may allow a reduction in the time required for dispersion and / or reduce the added amount of

chelating agents or proteolytic enzymes, especially trypsin. During the chemical or proteolytic dispersion step, the dispersion device according to the invention could be used continuously in recirculation or in a continuous flow mode. Another advantage is that the use of the dispersion device according to the invention during the chemical or proteolytic dispersion stage results in a better release of the microcarrier cells. This use increases the yield of the collected cells after the discarding of the microcarriers by clarification. At least one dispersion device can be used continuously in recirculation during cell culture. Depending on the type of cell, the history of the culture and the maintenance conditions, cell aggregates can be produced during the culture. It would be beneficial to disperse these aggregates into individual cells (that is, to increase the contact surface between the cells and the culture medium, to avoid the phenomena of apoptosis). In this particular situation, the dispersion device of the present invention can be used in recirculation since it allows closed circuit operation.

[0048] The devices and methods of the invention are suitable for use with a variety of

cells including, but not limited to, prokaryotic cells such as bacteria and particularly Escherichia 15 coli (E. coli), and eukaryotic cells such as, but not limited to, yeast, plant cells, and animal cells including insect cells and mammalian cells. The biological compounds of interest are RNAs, DNAs, viruses or phages, proteins. Animal cells particularly suitable for the use of the present invention include cells of human, primate, rodent, of porcine, bovine, canine, feline, sheep, avian origin and derivatives thereof. In general, animal cells include epithelial cells, which may be primary cells derived from a sample of embryonic tissue or a sample of adult tissue, such as keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells or retinal epithelial cells, or transformed cells or established cell lines (e.g., 293 human embryonic kidney cells, cervical HeLa epithelial cells or derivatives thereof (e.g., HeLaS3), retinal cells human PER.C6 and human keratinocytes HCAT), or derivatives thereof. The 25 cells can be normal cells, or they can be genetically altered. Other animal cells, such as CHO cells, COS cells, VERO cells, BHK cells (including BHK-21 cells), CLDK cells, CRFK cells, PK15 cells, MDBK cells, MDCK cells, TCF cells, TDF cells, CEF cells and derivatives thereof, are also suitable for the application of the present invention.

[0049] Cells are collected by any means known to persons skilled in the art,

including settlement or centrifugation. Cells can be collected and concentrated by centrifugation, in particular by cuvette centrifugation. In addition, cells can be stored refrigerated or frozen.

[0050] The invention will now be further described by means of the following non-limiting examples.

Example 1:

[0051] The preparation, propagation and infection of hen embryo cells with the Marek's disease virus (MDV) were carried out in rolling bottles of 1700 cm2. Chicken embryo cells

Infected from all the rolling bottles were dissociated by trypsinization after achieving an ECP (cytopathic effect) of 50 to 70%, then collected by centrifugation at 500g for 12 minutes.

[0052] After centrifugation, the supernatant was removed and freezing media were added to the sediment to obtain a cell suspension, but it contained many clearly visible aggregates at

simple sight

[0053] For dispersion of the aggregates, two procedures were used. In a first procedure, the cell suspension of 406 rolling bottles was homogenized by manual pipetting with a 50 ml pipette.

The resulting suspension was improvised filtered in a 800 pm nylon bag, adjusted in volume with some freezing media and then analyzed.

[0054] In parallel, the cell suspension equivalent to 48 rolling bottles was processed 8 times consecutively through a continuous flow dispersion device according to the invention and as illustrated

55 in Fig. 1.

[0055] The dispersion device constituted two portions, each having a conduit (with a cylindrical shape, 34 mm in length and an internal diameter of 36.5 mm) that ended with an obstacle (a sheet perpendicular to the axis of the conduit) ( see Fig. 1). Each sheet included three straight holes with a cylindrical shape

each having a diameter of 3 mm, and equidistant and parallel to the axis of the duct.

[0056] With one portion, the distance between a hole and the axis of the duct was 31 mm (see Fig. 2A). With the other portion, this distance was equal to 10 mm (see the model in Fig. 2B).

5

[0057] The two ducts joined together opposing the ends of the duct that had the obstacle in them at a distance between the two obstacles of approximately 6 mm).

[0058] The cell suspension recovered after centrifugation was injected through the dispersion device 10 with a flow rate of approximately 150 1 / h using a peristaltic pump. They were taken

samples before and after each step through the device and analyzed after filtration with 800 pm nylon. Injection through the dispersion device and sampling and analysis were reproduced 7 times.

[0059] Samples were analyzed for individual cell quantities by visual cell count with a Thomas cell, for the viability percentage by FACS (classification of activated cells

by fluorescence), and for the presence of aggregates by visual observation of the filtration residues. These results are presented in table 1.

Table 1:

 Sampling

 Individual cell count (cells / ml) Viability (%) Visual observations Viral titer (Log10ufp / ml)

 1st step Before filtration

 3.0E07 80 Very thick Very large lumps 6.99

 1st step After filtration

 4.4E07 84 Many lumps Many residues in the filter 7.05

 2nd step After filtration

 3.8E07 77 Many lumps Many residues in the filter 7.16

 3rd step After filtration

 4.3E07 75 Some lumps Less waste on the filter 6.83

 4th step After filtration

 5.2E07 71 Less lumpy few residues in the filter 7.14

 5th step After filtration

 5.8E07 70 Very few lumps few residues in the filter 6.95

 6th step After filtration

 4.9E07 68 Very few lumps few residues in the filter 7.17

 7th step After filtration

 4.8E07 66 Very few lumps few residues in the filter 7.1

 8th step Before filtration

 4.4E07 68 Few lumps 6.9

 Step 8 After filtration

 6.2E07 69 Very few lumps few residues in the filter 6.95

 Manual division After filtration

 4.1E07 63 Very few lumps Few residues in the filter 7.02

twenty

[0060] These results show that each step of the culture suspension containing cell aggregates through the continuous flow dispersion device according to the present invention had an effect on cell aggregates. The viability evaluated by FACS analyzes decreased with the number of steps, while the number of lumps visible to the naked eye decreased and the viral titer remained stable. With this kind of

25 cell culture (with chicken embryo cells infected with MDV) there was an optimal result for the use of the device for dispersion of aggregates with five steps.

[0061] These results also show that in-line dispersion of infected cells through a continuous flow dispersion device of the invention results in the dispersion of many aggregates, by

30 increased performance of the individual cells, and the preservation of more viability compared to when manual pipetting is used.

Example 2:

[0062] The preparation, propagation and infection of hen embryo cells with Marek's disease virus (MDV) were carried out in rolling bottles of 1700 cm2. Infected hen embryo cells from all rolling bottles were dissociated by trypsinization after compliance with an ECP (cytopathic effect) of 50 to 70%, then collected by centrifugation at 500g for 10 minutes. Be

5 processed around 2300 rolling bottles.

[0063] After centrifugation, the supernatant was removed and freezing media were added to the centrifuge bowl containing the sediment. The cuvette was turned by hand to obtain a thick cell suspension, but it still contained many aggregates visible to the naked eye. The suspension of

10 cells were removed for 10 minutes and divided equally into six packages.

[0064] A package was processed as standard without cell division. The cell suspension was filtered directly on a cheesecloth. The filtrate was further diluted with freezing media before filling.

[0065] The remaining five packages were divided continuously before filtering with cheesecloth and the

dilution with freezing media. For division, the number of dispersion devices (dispersion devices as described in Example 1) and the processing flow rate were changed from one serial arrangement to the other. The final products were analyzed for the cell count expressed in the number of cells per rolling bottles for a comparison of tests. The results are presented in Table 2.

twenty

Table 2:

 Split conditions

 Number of processed rolling bottles Individual cell count (millions of rolling cells / bottles)

 Standard

 388 423

 3 serial dispersion devices 50 l / h

 388 466

 7 serial dispersion devices 50 l / h

 388 452

 5 serial dispersion devices 150 l / h

 388 466

 3 serial dispersion devices 250 l / h

 367 431

 7 serial dispersion devices 250 l / h

 388 436

[0066] These results show that the processing of the culture suspension containing cell aggregates through various devices according to the present invention in series had an effect on the amounts

25 individual cells recovered at the end of the process.

[0067] These results also show that the number of series continuous flow dispersion device elements and the processing flow rate had an impact. Optimum results were obtained for a processing flow rate of 50 l / h and 3 serial dispersion devices, and for a flow rate of

30 150 l / h processing and 5 serial dispersion devices.

Claims (12)

1. A continuous flow dispersion device for dispersion of aggregates, the device comprising an upstream entrance at one end of a conduit, at least two obstacles having 5 opposing surfaces, said obstacles being a first or upstream entry obstacle disposed within the conduit, said upstream entry obstacle being perforated by at least one transverse hole and obstructing from about 50% to about 99.9% of the area of internal cross-section of the conduit, a second or downstream exit obstacle disposed within the conduit and a downstream exit obstacle being perforated by at least one transverse hole and obstructing from approximately 50% to 10 approximately 99.9% of the internal cross-sectional area of the conduit, where the longitudinal axis of any transverse orifice of any of the obstacles does not align with the longitudinal axis of any other orifice of an anterior or posterior obstacle, a turbulence chamber defined by the opposite surfaces of the first and second obstacles and the inner wall of the duct between said obstacle os, and where the distance between two successive obstacles is about 0.1 to about 10 times the smallest hole diameter of any of two successive obstacles, and an outlet downstream at the opposite end of the conduit; and wherein the entrance to the device conduit and the upstream entry obstacle form a first portion of the device, and the exit of the device conduit and the downstream exit obstacle form a second portion of the device and wherein the first and first portions second, they are separable, wherein the device further comprises a spacer seal; wherein, in addition, the conduit in each of the first and second portions 20 comprises a longitudinal light, in which the first and second portions can be juxtaposed and secured to each other such that a turbulence chamber is defined by the at least two obstacles and at least one inner wall of the spacer seal. 2. The device according to claim 1, wherein the entry and exit obstacles are removable from the device or portions thereof. 3. The device according to claim 1, wherein the upstream entry obstacle and the downstream exit obstacle are perpendicular to the direction of fluid flow through the conduit. The device according to claim 1, wherein a hole or holes that cross the obstacle of Upstream entry and the downstream exit obstacle are parallel to the longitudinal axis of the duct. 5. The device according to claim 1, further comprising a securing means for maintaining the integrity of the device. 35 6. The device according to claim 1, wherein the entry duct, the entry and exit obstacles, the turbulence chamber and the exit duct are formed as a single integral unit. 7. The device according to claim 1, further comprising at least one additional obstacle 40 thereby defining at least two turbulence chambers. 8. The device according to claim 1, wherein each of the upstream and downstream entry obstacles comprises three transverse holes, wherein the cross-sectional configurations of the holes are selected from circular, ovoid, concentric and rectilinear, creating each of 45 that way an obstruction of approximately 50% to approximately 99.9% of the cross section of the conduit, and where the holes may be identical or different from each other, and where the longitudinal axis of any hole a through the first obstacle it does not align with the longitudinal axis of any hole of the second obstacle. The device according to claim 1 comprising an upstream inlet at one end of a conduit, at least two obstacles having opposite surfaces, said obstacles being a first or upstream entry obstacle within the conduit, the upstream entry obstacle being perforated by at least one transverse hole and obstructing approximately 50% to approximately 99.9% of the internal cross-sectional area of the conduit, a second or downstream exit obstacle inside the conduit, and the downstream exit obstacle being perforated by at least one transverse hole and obstructing approximately the 50% to approximately 99.9% of the internal cross-sectional area of the conduit, where the upstream entry obstacle and the downstream exit obstacle are perpendicular to the direction of fluid flow through the conduit and the holes that they cross the entry obstacle and the exit obstacle downstream are parallel to the longitudinal axis of the duct, and where the longitudinal axis of any hole transverse of any of the obstacles does not align with the longitudinal axis of any other hole of an anterior or posterior obstacle, and a turbulence chamber defined by the opposite surfaces of the first and second obstacles and the internal wall of the conduit between said obstacles, and wherein the distance between two successive obstacles is about 0.1 to about 10 times the diameter of the smallest hole of any two of two successive obstacles, and an outlet downstream at the opposite end of the conduit, where the entrance to the duct of the device and the upstream entry obstacle form a first portion of the device, and the outlet of the device duct and the downstream exit obstacle form a second portion of the device and wherein the first and second portions are separable, and a spacer seal, where the turbulence chamber is defined by the opposite surfaces of the two obst asses and the inner wall 10 between obstacles device. 10. The device according to claim 9, wherein the conduit is cylindrical in shape, the entry obstacle has 3 holes that create an obstruction of approximately 98% of the cross section of the conduit; the exit obstacle has 3 holes that create an obstruction of approximately 98% of the 15 cross section of the duct, where the holes of the exit obstacle do not have the same alignment longitudinal with respect to any hole of the entry obstacle, and where the distance between these two Obstacles is approximately twice the diameter of any hole. 11. A method for dispersion of aggregates, comprising the steps of: twenty (1) passing a cell suspension containing cell aggregates through at least one device according to Claim 1; (2) dissociate the cell aggregates within the cell suspension, (3) optionally repeat step (1) by passing the suspension through the device again; Y 25 (4) collect the cell suspension containing the aggregates of disjointed cells. 12. The method according to claim 11, wherein the cell suspension is passed through a serial arrangement of a plurality of devices according to Claim 1. 30 13. A cell culture procedure, comprising the steps of: (1) introduce cells to grow in a culture medium and grow the cells; (2) displace or suspend cultured cells in a medium, wherein the suspension comprises aggregates of cells; (3) passing the cell suspension containing cell aggregates through a continuous flow dispersion device according to claim 1 or through a serial arrangement of such devices, thereby disintegrating the cell aggregates and releasing cells individual of them; (4) optionally repeat steps from (1) to (3) are repeated as many times as necessary, and (5) reintroduce at least a portion of the disjointed cell suspension from step (3) to the culture batch and reculture the cells. 14. The method according to claim 13, wherein the culture medium comprises microcarriers. 15. The method according to claim 13, wherein step (3) is continuous during the period of cultivation.